Continuous process for ethanol production by bacterial fermentation using pH control

ABSTRACT

A continuous process for the production of ethanol by fermentation of an organism of the genus Zymomonas is provided. The method is carried out by cultivating the organism under substantially steady state, anaerobic conditions and under conditions in which ethanol production is substantially uncoupled from cell growth. By controlling pH in the fermentation medium between a pH of about 3.8 and a pH less than 4.5, it is possible to optimize kinetic parameters, such as qs (g substrate/g biomass-hr-1) and qp (g ethanol/g biomass-hr-1) without adversely affecting ethanol concentration in the fermentation medium.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.772,492, filed Apr. 12, 1985, the entire disclosure of which is reliedupon and incorporated by reference herein.

BACKGROUND OF THE INVENTION

This invention relates to the bioconversion of a substrate by bacterialfermentation, and more particularly, to a continuous process for theproduction of ethanol by fermentation using strains of Zymomonasbacteria.

Bacterial ethanol fermentation has been known in the art for many years,and in recent years fermentation using strains of Zymomonas mobilis hasreceived increasing attention. The Z. mobilis strains convert a suitablesubstrate, such as glucose or another sugar, to ethanol. Significantlyhigher specific rates of sugar uptake and ethanol production andimproved yield compared to traditional yeast fermentation have beenreported for these Zymomonas strains.

Fermentation by Z. mobilis has been carried out in batch and continuousculture. The fermentation product (ethanol) is dissolved in the liquidmedium in the fermenter. The liquid medium is separated from solids(chiefly biomass) before the ethanol is recovered. Separation of thesetwo phases early in the product-recovery process train is required.Before such a fermentation achieves commercial acceptance, however,productivity must be improved. The reported efforts to date have focusedon developing more productive bacterial strains and modifying theconfiguration of the fermenter used in the fermentation. For example,improvement in ethanol productivity using a continuous culture with acell recycle system has been reported with Z. mobilis strains.

Recovery of the fermentation product can be a complex and multifacetedtask. A significant proportion of the overall cost in a fermentationplant often must be spent for ethanol recovery. A recently reportedtechnique involves the use of a flocculent strain of Z. mobilis thatsettles in the fermenter allowing the supernatant containing the ethanolto be withdrawn while leaving a majority of the cells in the fermenter.This method is based on the well-known gravity sedimentation principlefor separating liquids and solids. The more conventional approaches forseparating fermentation broth from biomass involve the withdrawal of aportion of the culture medium from the fermenter and separation of thetwo phases by centrifugation or filtration techniques. Regardless of thetechnique employed, for a particular ethanol recovery process, it isdesirable to reduce the quantity of biomass in order to reduce the loadof solids on the sedimentation, centrifugation or filtration apparatus.

At the same time, however, the yield of ethanol from the fermentationmust be maximized. Since product formation cannot occur in the absenceof biomass, ethanol formation is dependent on cell mass concentration.In fact, the rate of ethanol production in the fermenter is directlyproportional to the quantity of biomass in the fermenter under steadystate conditions. Thus, within the limits of the metabolic regulatorycontrols of the microorganism and process dynamics, increasing thebiomass in the fermenter while maintaining other conditions constantwill shorten the time required to produce a given amount of ethanol.However, this seemingly simple approach for optimizing processperformance will have an adverse effect on the ethanol recovery processbecause the load of solids on the separating equipment will becorrespondingly increased.

It is well known that the substrate, such as glucose, is the largestitem of raw material cost in the fermentation. Therefore, the presenceof substrate in the effluent from the fermenter in a continuousfermentation is to be avoided. The continuous fermentation should beconducted at optimum process product yield, which occurs when thesubstrate is completely converted to ethanol and when the substrate isminimally diverted from product (ethanol) formation to cell masssynthesis (i.e., when the growth yield with respect to carbon substrateis minimized).

Thus, there exists a need in the art for a continuous process for theproduction of ethanol using strains of Zymomonas in which the substratefed to the fermenter is converted to ethanol in as short a time aspossible. The process should permit a reduction in the quantity ofbiomass in the fermenter in order to obtain a corresponding reduction inthe load of solids on the ethanol recovery apparatus. In addition, thequantity of substrate in the effluent from the fermenter should beminimized.

SUMMARY OF THE INVENTION

This invention aids in fulfilling these needs in the art by providing acontinuous process for the production of ethanol, wherein pH in theferementation medium is monitored and controlled in order to optimizekinetic parameters, such as the specific rate of substrate uptake(q_(s)) and the specific rate of ethanol formation (q_(p)), withoutadversely affecting concentration of ethanol in the medium. Moreparticularly, this invention provides a continuous process for theproduction of ethanol, wherein the process comprises feeding an aqueoussubstrate solution substantially continuously to a reactor containing afermentation medium and a submerged culture of an organism of genusZymomonas. The organism is cultivated to produce ethanol undersubstantially steady state, anaerobic conditions in an aqueous nutrientmedium containing assimilable carbon, nitrogen and phosphorus, andoptionally, other salts as needed. Cultivation is carried out under suchconditions that ethanol production is substantially uncoupled from cellgrowth. The pH in the fermentation medium is controlled between a pH ofabout 3.8 and a pH of less than 4.5, preferably at a pH of about 4.0 toabout 4.2. An effluent containing ethanol can be removed from thereactor.

When the pH in the fermentation medium is controlled according to thisinvention, the biomass expresses its maximum value for both specificrate of substrate uptake (q_(s)) and specific rate of product formation(q_(p)) at any given value of dilution rate (D). The process of thisinvention can be carried out with or without nutrient-limitingconditions. The limiting nutrient is either nitrogen, potassium orphosphorus, when nutrient-limiting conditions are employed. Theimposition of nutrient limitation makes it possible to conduct thefermentation at a lower biomass concentration at a given substrateconcentration in the feed stream to the fermenter or at a given dilutionrate than under conditions of nutrient-excess.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be more fully described with reference to thedrawings in which:

FIG. 1 is a schematic drawing of fermentation apparatus of the type thatcan be employed in practicing the process of this invention;

FIG. 2 is a graph showing the effect of pH on the specific growth rate(u) in continuous chemostat culture using Z. mobilis strain ATCC 29191;

FIG. 3 is a graph showing the effect of pH on the specific rate ofsubstrate uptake (q_(s)) and the specific rate of product formation(q_(p)) in the continuous chemostat culture using Z. mobilis strain ATCC29191;

FIG. 4 is a graph showing the effect of pH on the growth yieldcoefficient (Y_(x/s)) for the same fermentation; and

FIG. 5 is a graph showing the effect of pH on concentration of ethanol([P]) in the medium of the same fermentation.

DETAILED DESCRIPTION

Ethanol production by yeast is normally a growth related process.Maintenance metabolism by non-growing yeast accounts for some alcoholproduction reasonably late in the fermentation when the concentration ofethanol is relatively high, but the rate is usually very slow. Ethanoland carbon dioxide are by-products of energy metabolism and they areproduced in response to the energy demands of the various biosyntheticand energy-requiring processes (including growth) of the yeast cell.Thus, in yeast, there exists a fairly tight coupling between anabolicand catabolic processes. As a consequence of this well-regulated andconservative metabolism, very high concentrations of yeast are needed inorder to achieve an acceptable rate of ferementation bynon-proliferating yeast.

This is in direct contrast to the situation which exists in Zymomonasfermentation where energy supply is not controlled by the energy demandof the biosynthetic or anabolic processes of the organism. Theefficiency of energy utilization appears to be capable of varyingbetween a condition of complete uncoupling, as in the case of glucosemetabolism by washed cell suspensions, to a condition of maximalutilization associated with growth in an optimized environment. Thus,the fermentation process can be directed towards either maximum energyconservation, such as would be desirable for cell mass production (e.g.,starter cultures/inoculum), or it can be directed towards maximum energyuncoupling, such as that which occurs during ethanol production withminimal or no substantial cell growth.

The growth yield coefficient (Y_(x/s)) is a direct reflection of theefficiency with which the energy supplied by glucose metabolism isutilized during growth. A high growth yield coefficient is indicative oftight energetic coupling. Energetic uncoupling is manifested by areduced growth yield coefficient and an increase in the specific rate ofethanol formation (q_(p)).

It has now been found that the specific rate of ethanol formation can beincreased by controlling pH during the fermentation process.Specifically, the rate of ethanol production by a Zymomonas cultureunder steady state conditions can be optimized by carrying out thefermentation under controlled conditions at a relatively low pH. Thisresult is apparently due to the fact that pH control induces energeticuncoupling such that energy conservation is minimized and energydissipation is maximized. More particularly, the production of ethanolin the process of this invention is carried out under conditions ofuncoupled growth in a fermentation medium between a pH of about 3.8 anda pH less than 4.5.

Conventional expressions are used throughout this description when thekinetics of the fermentation process are discussed. The abbreviationsidentified in the following list have been used in order to facilitatethe discussion. Units of measure have been included where appropriate.

S_(r) =Substrate concentration in the feed stream to the fermenter;g/liter, i.e. g/L.

S_(o) =Substrate concentration in the effluent from the fermenter; g/L.

V=Volume of fermentation medium in the fermenter; L.

X=Concentration of biomass in the fermentation medium (dry basis); g/L.

[P]=Concentration of ethanol in the fermentation medium; g/L.

u=Specific growth rate (mass); hr⁻¹.

D=Dilution rate; hr⁻¹.

N*=Amount of a given nutrient; g.

q_(s) =Specific rate of substrate uptake; g substrate/g biomass-hr⁻¹.

q_(s) ^(max) =Maximum observed specific rate of substrate uptake; gsubstrate/g cell-hr⁻¹.

q_(p) =Specific rate of ethanol formation; g ethanol/g biomass-hr⁻¹.

q_(p) ^(max) =Maximum observed specific rate of ethanol formation; gethanol/g cell-hr⁻¹.

VP=Volumetric Productivity; g ethanol/g cell-hr⁻¹.

Y_(n) =Growth yield coefficient for a specified limiting nutrient, n; gdry biomass/g-atom nutrient.

n=Limiting nutrient, i.e., nitrogen, potassium or phosphorus.

Y_(p/s) =Product Yield Coefficient=q_(p) /q_(s) ; g ethanol produced/gsubstrate consumed.

Y_(x/s) =Growth Yield Coefficient; g biomass/g-atom of substrateconsumed.

YE=Yeast extract (Difco) in aqueous medium; g/L.

AC=High grade anhydrous NH₄ Cl in aqueous solution; g/L.

AS=High grade anhydrous (NH₄)₂ SO₄ in aqueous solution; g/L.

Eth=Ethanol.

Glu=Glucose.

As used herein, the term "uncoupled growth" means that the specific rateof substrate uptake (q_(s)) is substantially independent of specificgrowth rate (u). As energetic uncoupling occurs, the growth yieldcoefficient (Y_(x/s)) and the specific growth rate (u) decrease, whereasthe specific rate of substrate uptake (q_(s)) and the specific rate ofproduct formation (q_(p)) each increase, eventually reaching maximalvalues and remaining constant when energetic uncoupling is complete.Whereas in batch culture, uncoupled growth is recognized as a decreasein u_(max) independent of q_(s), in continuous culture, where D(equivalent to u) is fixed at a value less than u_(max), one observes adecrease in Y_(x/s) and an increase in q_(s) with an increase inmaintenance energy coefficient (m_(e)). By controlling pH in thefermentation medium when biomass concentration is substantiallyconstant, it is thus possible to optimize kinetic parameters, such asthe specific rate of substrate uptake (q_(s)) and the specific rate ofethanol formation (q_(p)), without adversely affecting concentration ofethanol ([P]) in the fermentation medium.

As used herein, the expression "nutrient-limited fermentation" andsimilar expressions mean a fermentation of an organic substrate by aZymomonas strain, where the fermentation is carried out in a continuousprocess under steady-state conditions in a medium in which one of morenutrients are present in an amount such that the rate of growth islimited by the availability of one or more of the essential nutrients.

As used herein, the expression "limiting nutrient" means nitrogen,potassium or phosphorus.

The process of this invention is carried out as a continuousfermentation. The term "continuous" is used in its conventional senseand means that nutrients are fed to a fermenter substantiallycontinuously and that an output, or effluent, stream is substantiallyconstantly withdrawn from the fermenter. The nutrient stream usuallycomprises an aqueous organic substrate solution. The effluent streamcomprises biomass and the liquid phase from the fermentation medium.

FIG. 1 will provide a background for the following discussion in whichthe method and apparatus for carrying out this invention are describedin detail. Referring to FIG. 1, a nutrient medium 1 in reservoir 2 isfed by a pump 3 to a fermenter 4 containing a fermentation medium 5. Themedium is maintained at a constant volume in the fermenter by means ofan overflow weir 6 that empties into a container 7. Carbon dioxideformed during the fermentation is vented at 8.

The fermenter 4 is provided with an agitator 9 for mixing the fermentercontents. A pH probe 10 is immersed in the fermentation medium 5 and isconnected to a pH controller 11 for regulating the amount of pHregulating agent 12 in reservoir 13 added by a pump 14 to thefermentation medium 5.

The temperature of the fermentation medium is monitored by temperatureprobe 15. The fermentation medium is heated or cooled as requiredthrough a coil 16 and temperature is regulated by the temperaturecontroller 17.

The fermentation medium can be formulated so that all but a singleessential nutrient are available in excess of the amount required tosynthesize a desired cell concentration. The single growth-limitingnutrient controls the size of the steady-state cell population.

Effect of pH on Fermentation Kinetics and Yield Coefficient

The evolution of energy during the fermentation process is related tothe metabolic cycle of the Zymomonas organism. When the carbon substrateis being actively incorporated into cell mass during the growth phase,most of the energy generated in the process is conserved in the biomass.It was discovered that by lowering the pH in the fermentation medium andmaintaining the lowered pH, catabolism of the carbon source becomesdissociated from cell growth, the growth yield coefficient decreases andthere is increased production of ethanol. Thus, pH control can be usedto promote uncoupling and to regulate metabolism in continuous culture.It was also found that the use of pH control makes it possible toincrease the kinetics of product formation without adversely affectingproduct concentration in the fermentation medium.

The production of ethanol in continuous Zymomonas culture can be carriedout in two stages. In the first stage, cell growth is carried out in thepresence of suitable nutrients and a suitable carbon source and at a pHof about 5.5 to about 6.5, preferrably at a pH of about 5.7. Thefermentation is continued in the second stage between a pH of about 3.8and a pH less than 4.5. Cell yield can be maximized in the first stageand the rate of ethanol production can be maximized in the second stageas will be apparent from FIGS. 2, 3, 4 and the following description.

Z. mobilis strain ATCC 29191 was grown in continuous, steady-statechemostat culture using the apparatus described in Example 1. Thefermentation medium contained 5% (w/v) D-glucose as the fermentablecarbon source and as growth-limiting nutrient, and 0.5% yeast extract(30 mM NH₄ Cl) plus other inorganic salts (see TABLE 7). The dilutionrate was held constant at 0.15 hr⁻¹, and the temperature was controlledat 30° C. The fermentation medium was sparged with nitrogen gas. The pHwas monitored and controlled by addition to the fermentation medium ofan aqueous solution containing 3N potassium hydroxide. The pH was variedover the range 4.0 to 7.0 and glucose concentration, biomassconcentration (X) and ethanol concentration ([P]) were determined asdescribed in Example 1. The specific rate of glucose uptake (q_(s)), thespecific rate of ethanol formation (q_(p)), the specific growth rate (u)and the growth yield coefficient (Y_(x/s)) were calculated. The resultsare plotted in FIGS. 2, 3, and 4.

FIG. 2 shows the pronounced effect that pH has on the specific growthrate (u). The specific growth rate increased to a maximal value when thepH in the fermentation medium was increased from a pH of 4 to a pH ofabout 6.5 and then rapidly declined. At higher pH values, the specificgrowth rate declined. These results indicate that optimum pH values forgrowth are about 5.5 to about 6.5 for Z. mobilis strain ATCC 29191 underthe stated conditions.

The data in FIG. 4 shows that growth yield coefficient (Y_(x/s)) in acontinuous (5% glucose) fermentation with the growth rate fixed at 0.15hr⁻¹ reached a maximal value at a pH of about 6.0 and declined withdecreasing pH in the fermentation medium. On the other hand, referringto FIG. 3, the specific rate of substrate uptake (q_(s)) and thespecific rate of product formation (q_(p)) exhibited oppositetrends--q_(s) and q_(p) each increased with decreasing pH and reachedoptimal levels at a pH values of less than 4.5. The specific rate ofethanol formation (q_(p)) exhibited a trend similar to q_(s), since theproduct yield coefficient (Y_(p/s)) was substantially constant duringthe fermentation and q_(s) is proportional to q_(p) under thesecircumstances. These results confirm that the overall fermentationprocess can be favorably influenced by carrying out cell growth atrelatively high pH in one stage and then effecting product enrichment(i.e., ethanol production) in a second stage at a lower pH.

FIG. 5 shows that ethanol concentration in the fermentation medium isnot significantly affected by pH in the range of 4 to 5; since ethanolconcentration remained constant over this pH range. Although it appearsthat pH does not affect ethanol concentration over the pH range of 4 to5, it was observed that at a pH of 7, q_(s) increased several fold andY_(x/s) correspondingly decreased, but ethanol concentration decreasedmarkedly. These results suggest that, under the described fermentationconditions using the Z. mobilis strain ATCC 29191 at a pH of 7, glucosewas being metabolized by the cells at a very fast rate, but the glucosewas not being converted to the desired product, ethanol.

This work shows the advantage of controlling pH in the fermentationprocess such that one either achieves maximal energy coupling for thepurpose of maximizing cell mass production, or maximal energeticuncoupling for the purpose of maximizing the specific rate of ethanolproduction and product yield (minimizing cell mass production). Maximalenergy coupling in continuous culture can be obtained at a pH of about5.5 to about 6.5, preferrably at pH of about 5.7. Maximal energyuncoupling can be obtained between a pH of about 3.8 and a pH less than4.5, preferably at a pH of about 4.0 to 4.2. Ethanol production in thefermentation process of this invention is thus achieved within a pHrange that is significantly less than pH value that is optimum for cellgrowth. Less of the substrate is diverted to cell mass productionleaving more of the substrate available for ethanol production. A moredetailed description of the procedure for monitoring and controlling pHfollows.

The pH in the culture medium generally falls during the course offermentation. The pH is measured in the medium containing varioussolutes, including the nutrients required for cell metabolism. The pHcan be intermittently or continuously monitored using conventionalinstruments that automatically measure and regulate pH. For example, pHcan be measured with any of the well-known immersion units orflow-through units, and the resulting measurement can be coupled to acommercially available recorder, controller and reagent feeder. The pHmeasuring device should be calibrated to the same temperature at whichthe fermentation is operated.

Acidic or basic substances can be added as pH regulating agents toadjust pH during the course of fermentation. The regulating agent is asubstance that is non-toxic and substantially non-inhibiting to themicroorganism. The regulating agent can be in liquid, solid or slurryform, and mixtures of different substances can be employed. Solutions ofacidic and basic substances are preferred since they can be rapidlydispersed in the fermentation medium and can yield a more immediate pHchange in the medium. The regulating agent is typically a hydroxide oran organic or inorganic acid. Examples of suitable pH regulating agentsare potassium hydroxide, sodium hydroxide and hydrochloric acid.

Proper selection of the pH regulating agent can aid in ensuring properpH control. For example, in order to prevent wide cycling of pH, acorrective reagent that produces a small rate of change of pH near thecontrol point when a moderate volume of the reagent is used is preferredover an equivalent volume of a different reagent that produces a rapidrate of change of pH.

The culture medium can be buffered in order to restrict pH changesduring fermentation. A buffering agent that is non-toxic andsubstantially non-inhibiting to the microorganism can be employed forthis purpose. The formation of insoluble compounds or complexes andundesired side reactions in the medium should be avoided. An example ofa suitable buffer than can be employed in the pH ranges of thisinvention is phosphate. The optimum buffer type and buffer concentrationcan be determined with a minimum of experimentation.

When the fermentation is carried out under nutrient-limiting conditions,it is preferred to use a corrective reagent and a buffer, which are notionizable to the limiting nutrient in the culture medium. If thecorrective reagent or buffer ionize to form the limiting nutrient, itbecomes very difficult to control the concentration of the limitingnurient in the fermentation medium.

General Fermentation Conditions

Fermentation can be carried out in a bioreactor, such as a chemostat,tower fermenter or immobilized-cell bioreactor. Fermentation ispreferably carried out in a continuous-flow stirred tank reactor. Mixingcan be supplied by an impeller, agitator or other suitable means andshould be sufficiently vigorous that the vessel contents are ofsubstantially uniform composition, but not so vigorous that themicroorganism is disrupted or metabolism is inhibited.

Fermentation is carried out with a submerged culture and undersubstantially anaerobic conditions. While the invention is described inthe Examples hereinafter with freely mobile cells, it will be understoodthat immobilized cells can also be employed. The fermenter is preferablyenclosed and vented to allow the escape of carbon dioxide evolved duringthe fermentation. Oxygen at the surface of the fermentation medium is tobe avoided. This may inherently occur as the heavier carbon dioxideevolved during the fermentation displaces the oxygen in the gas phaseabove the medium. If necessary, the gas phase above the medium can bepurged with an inert gas to remove oxygen and maintain substantiallyanaerobic conditions.

The fermenter can be operated with or without cell recycle. Cell recyclemakes it possible to increase the productivity of the system byoperating at a higher steady-state cell concentration compared to asimilar system without cell recycle. When cell recycle is employed, aportion of the fermenter contents is withdrawn from the fermenter, theethanol-containing phase is separated from the effluent, and theresulting concentrated cells are returned to the fermenter. Theseparation is typically carried out by microfiltration orcentrifugation. Since the process of this invention is carried out atreduced biomass concentration in the fermenter, the load of solids onthe cell recycle apparatus is reduced and ease of ethanol recovery isincreased.

The composition of the effluent stream can vary and will usually be thesame as the composition of the fermentation medium. When a flocculentstrain of Zymomonas is employed, however, or if partial separation ofbiomass from the liquid phase otherwise occurs in the fermenter, theeffluent can contain a larger proportion of biomass or liquid phasedepending upon the location where the effluent is withdrawn from thefermenter.

The microorganism employed in the process of this invention is agram-negative, faculative anaerobic bacterium of the genus Zymomonascapable of fermenting an organic substrate to ethanol undersubstantially anaerobic continuous culture conditions. Typical strainsare Zymomonas mobilis and Zymomonas anaerobia. Suitable strains ofZymomonas are available from microorganism depositories and culturecollections. Examples of suitable Z. mobilis strains are thoseidentified as ATCC 10988, ATCC 29191, ATCC 31821 and ATCC 31823 [ex ATCC31821]. Examples of other strains of Z. mobilis are those identified asNRRL B-14023 [CP 4] and NRRL B-14022 [CP 3]. Flocculent strains can alsobe employed. These strains include ATCC 35001 [ex ATCC 29191], ATCC35000 [ex NRRL B-14023], ATCC 31822 [ex ATCC 31821] and NRRL B-12526 [exATCC 10988]. Z. mobilis strains are occasionally referred to in theliterature by the following alternate designations:

                  TABLE 1                                                         ______________________________________                                        Culture Collection                                                            Accession No.      Literature Designation                                     ______________________________________                                        ATCC 10988         Strain ZM 1                                                ATCC 29191         Strain ZM 6 (or Z6)                                        ATCC 31821         Strain ZM 4                                                ATCC 31822         Strain ZM 401                                              ATCC 31823         Strain ZM 481                                              NRRL B-14022       Strain CP3                                                 NRRL B-14023       Strain CP4                                                 ______________________________________                                    

It will be understood that other Zymomonas strains can be obtained byselective cultivation or mutation as well as by genetic engineeringtechniques to provide microorganisms with desired metabolic properties.

The substrate employed in the process of this invention is an organic,fermentable substrate for the Zymomonas strain. As the carbon source forboth the growth and fermentation stages of the process, variouscarbohydrates can be employed. Examples of suitable carbohydrates aresugars, such as glucose, fructose and sucrose; molasses; starchhydrolysates; and cellulose hydrolysates. Other suitable substrates willbe apparent to those skilled in the art. The organic substrate can beemployed either singly or in admixture with other organic substrates.

The substrate is fed to the fermenter in aqueous solution. Theconcentration of organic substrate in the fermentation medium willdepend upon the culture conditions. The substrate is employed in anamount sufficient for cell growth and product formation. Typically, theconcentration of fermentable substrate in the feed stream to thefermenter will be about 50 to about 200 g/L. It will be understood thatwhen S_(r) is high, D is low in order to obtain nearly completeutilization of fermentable substrate.

The flow rate of the substrate solution to the fermenter will dependupon the size and configuration of the fermenter, the amount of biomassin the fermenter and the rate at which substrate is consumed, and can bedetermined with a minimum of experimentation. The flow rate should bebelow the rate at which a substantial amount of substrate appears in theeffluent from the fermenter. Preferably, the flow rate of the substratesolution to the fermenter should be such that the effluent substrateconcentration is less than about 5% S_(r), and should be such that theeffluent is substantially free of substrate under optimum operatingconditions.

The process of this invention can be carried out over a moderate rangeof temperatures. The effects of temperature changes on fermenterperformance are discussed below, but generally speaking, the process ofthis invention is carried out at a temperature of about 27° C. to about37° C., preferably about 30° C.

The process of this invention is carried out under sufficiently sterileconditions to ensure cell viability and metabolism. This requirescareful selection of the microorganism, sterilization of the apparatusfor the fermentation and of the liquid and gaseous streams fed to thefermenter. Liquid streams can be sterilized by several means, includingradiation, filtration and heating. Small amounts of liquids containingsensitive vitamins and other complex molecules can be sterilized bypassage through microporous membranes. Heat-treatment processes arepreferred for sterilizing the substrate feed stream and can be carriedout by heating the stream in a batch or continuous flow vessel. Thetemperature must be high enough to kill essentially all organisms in thetotal holding time. Water utilized in the preparation of the substratesolution and in the preparation of the fermentation broth in thefermenter can be sterilized in a similar manner or by other conventionaltechniques.

After the fermenter has been inoculated with the Zymomonasmicroorganism, the quantity of biomass is multiplied. The growingculture is allowed to complete the lag phase and substantially theentire exponential phase of growth before flow to the fermenter isinitiated. The fermentation is allowed to proceed under substantiallysteady-state conditions with the continuous introduction of freshsubstrate and the continuous withdrawal of product from the fermenter.While product formation is not solely associated with growth, it will beunderstood that a portion of the substrate fed to the fermenter goesinto cell maintenance. Thus, in the case of direct conversion of glucoseto ethanol: 1 mole glucose → 2 moles ethanol+2 moles CO₂. The maximumconversion is 2 mole ethanol per mole glucose or 0.51 g ethanol/gglucose, but theoretical yield cannot be achieved in practice since someof the substrate goes into cell mass. The process of this invention iscarried out at a yield of at least about 80%, preferably at least about94%, of theoretical yield. In this context, "complete fermentation"means that greater than 95% of the sugar substrate has been converted toethanol product.

Viable cell concentration in the fermenter will depend upon severalfactors, such as dilution rate, substrate concentration, maximum growthrate and growth yield coefficient. The fermenter can be operated over arange of biomass concentrations and the optimum concentration can bedetermined without undue experimentation. The practical range of valueswill generally depend upon process economics. For example, a continuouschemostat culture without cell recycle at maximum substrateconcentration in the feed stream can typically be operated at a maximumbiomass concentration (DWB) of about 3.5 g/L. A practical range ofbiomass concentrations is about 0.8 to about 3.2 g/L.

The concentration of ethanol in the fermentation medium should bemaximized in order to reduce the cost of product recovery. The processof this invention is carried out at ethanol concentrations up to about85 g/L, preferably about 45 g/L to about 85 g/L in the fermentationmedium.

Z. mobilis is sensitive to ethanol concentration, and at concentrationsin excess of about 50 g/L (5% w/v, at T≦33° C.), cell growth andmetabolism are retarded. This can be caused by a high concentration ofsubstrate in the feed stream to the fermenter. Thus, as the value forS_(r) is increased, the maximum dilution rate for substantially completeconversion of substrate to ethanol decreases. The process of thisinvention is carried out at a dilution rate of about 0.05 hr⁻¹ to about0.35 hr⁻¹, preferably about 0.1 hr⁻¹ to about 0.2 hr⁻¹.

These features of this invention will be more fully understood from thefollowing discussion. The effects of the limiting nutrient on fermenterperformance at various concentrations of substrate in the feed streamand different dilution rates are summarized below. The results obtainedin a series of experiments with varying operating conditions arereported in the following Tables.

Nitrogen-Limited Fermentation

Table 2 shows the effect of increasing the concentration of glucose inthe feed stream on the performance of a continuous fermentation by Z.mobilis strain ATCC 29191 in a chemostat at a constant dilution rate of0.15 hr⁻¹ under either nitrogen-limiting conditions or conditions ofnitrogen-excess. The amount of assimilable nitrogen was varied bychanging the amount of either yeast extract (YE), ammonium chloride (AC)or ammonium sulphate (AS). The amount of the nitrogen-limiting additivewas the minimal amount required to achieve maximal rate of sugarutilization and ethanol production by the Z. mobilis strain under thefermentation conditions.

                  TABLE 2                                                         ______________________________________                                        Amount of Assimilable Nitrogen as Either                                      Yeast Extract (Difco), Ammonium Chloride or                                   Ammonium Sulphate Required to Achieve Maximal Rate of                         Sugar Utilization and Ethanol Production by                                   Z. mobilis ATCC 29191 in Continuous Culture at                                Fixed Dilution Rate (0.15 hr.sup.-1) as a                                     Function of Feed Sugar Concentration (S.sub.r).                               ______________________________________                                        Excess Nitrogen                                                               S.sub.r   [P]    X                                                            g/L       g/L    g/L          q.sub.s                                                                            q.sub.p                                    ______________________________________                                        20        9.6    0.58         5.2  2.5                                        60        28     1.73         5.2  2.5                                        110       52     3.17         5.2  2.5                                        ______________________________________                                        Nitrogen Limitation                                                           S.sub.r                                                                             [P]     X                  YE    AC    AS                               g/L   g/L     g/L     q.sub.s                                                                            q.sub.p                                                                             g/L   g/L   g/L                              ______________________________________                                        20    9.6      0.36   8.3  3.9   0.8   0.19  0.23                             60    28      1.1     8.3  3.9   2.4   0.59  0.72                             110   52      2.0     8.3  3.9   4.4   1.10  1.32                             ______________________________________                                         Units: q.sub.s = g glu/g cellhr.sup.-1 ; q.sub.p = g eth/g cellhr.sup.-1.

The data in Table 2 show that a fermenter can be operated according tothis invention at a lower biomass concentration than undernitrogen-excess at a given glucose concentration. For example, with aglucose concentration (S_(r)) of 20 g/L in the feed stream, the biomassconcentration (X) in the fermenter was only 0.36 g/L undernitrogen-limiting conditions, whereas the biomass concentration was 0.58g/L under conditions of nitrogen-excess. A similar comparison can bemade for the other glucose concentrations shown in Table 2.

The data in Table 2 also show that the fermenter can be operated at ahigher specific rate of product formation (q_(p)) undernitrogen-limiting conditions than under conditions of nitrogen-excess ata given glucose concentration. The observed specific rate of productformation of 3.9 under nitrogen-limiting conditions was approximately1.56 times greater than observed q_(p) under condition ofnitrogen-excess.

In addition, the data in Table 2 demonstrate that as the concentrationof glucose in the feed stream (S_(r)) is increased, biomassconcentration (X) also increases under both nitrogen-limiting andnitrogen-excess conditions, but the biomass level is less undernitrogen-limiting conditions than under conditions of nitrogen-excessfor a given S_(r).

For near complete conversion of glucose to ethanol, the amount oflimiting nitrogen was increased as the glucose concentration wasincreased. For example, in order to achieve complete fermentation ofadded substrate, the quantity of yeast extract in the nutrient mediumwas increased from 0.8 g/L to 4.4 g/L when the glucose concentration inthe feed stream was increased from 20 g/L to 110 g/L. The cultureexpressed maximal values for q_(p) and q_(s) under these conditions.These results were entirely unexpected.

The data in Table 2 also show that nitrogen-limitation in thefermentation medium does not appreciably affect product yield whencompared with a similar fermentation carried out under conditions ofnitrogen-excess. The product yield was about 92% of the theoreticalmaximum yield in all cases. [[e.g., [28/(60×0.51)]×100=92%]]. Theseresults were also entirely unexpected.

Another series of fermentations similar to those summarized in Table 2was carried out, except that the concentration of glucose in the feedstream (S_(r)) to the fermenter was maintained at 110 g/L while thedilution rate (D) was varied. Table 3 shows the amount of assimilablenitrogen as either yeast extract (YE), ammonium chloride (AC) orammonium sulphate (AS) required to achieve maximal rate of sugarutilization and ethanol production under nitrogen-limiting conditionsand conditions of nitrogen-excess.

                  TABLE 3                                                         ______________________________________                                         Amount of Assimilable Nitrogen as either Yeast                               Extract (Difco) Ammonium Chloride or Ammonium Sulphate                        Required to Achieve Maximal Rate of Sugar Utilization                         and Ethanol Production by Z. mobilis ATCC 29191 in                            Continuous Culture at Fixed Feed Glucose                                      Concentration (S.sub.r = 110 g/L) as a                                        Function of the Dilution Rate (D)                                             ______________________________________                                        Excess Nitrogen                                                               D         S.sub.r                                                                              X                                                            hr.sup.-1 g/L    g/L          q.sub.s                                                                            q.sub.p                                    ______________________________________                                        0.1       110    2.53         4.4  2.1                                         0.15     110    3.17         5.2  2.5                                        0.2       110    3.63         6.1  2.9                                        ______________________________________                                        Nitrogen Limitation                                                           D                              YE    AC   AS    S.sub.o                       hr.sup.-1                                                                          S.sub.r X      q.sub.s                                                                             q.sub.p                                                                            g/L   g/L  g/L   g/L                           ______________________________________                                        0.1  110     1.33   8.3   3.9  3.0   0.71 0.88  5                              0.15                                                                              110     2.00   8.3   3.9  4.4   1.07 1.32  5                             0.2  110     2.65   8.3   3.9  5.9   1.42 1.74  5                             0.2  110     2.00   8.3              1.07 30                                  ______________________________________                                         Units: q.sub.s = g glu/g cellhr.sup.-1 ; q.sub.p = g eth/g cellhr.sup.-1.

The data in Table 3 demonstrate that a fermenter can be operatedaccording to this invention at a lower biomass concentration undernutrient limiting conditions than under conditions of nitrogen-excess ata given dilution rate. The fermenter can be operated at a higherspecific rate of product formation under nitrogen-limiting conditionsthan under conditions of nitrogen-excess at a given dilution rate. Asthe dilution rate is increased, biomass concentration in the fermenteralso increases with increasing amounts of nitrogen, but the biomasslevel is less under nitrogen-limitation than under nitrogen-excess.

The amount of assimilable nitrogen required for nitrogen-limitedfermentation is proportional to the dilution rate and must be increasedas the dilution rate is increased to achieve complete substrateconversion to ethanol. The significance of this feature of the inventioncan be more fully appreciated by comparing the data in Table 3. When thefermentation was carried out under nitrogen-limiting conditions and thedilution rate was doubled, say from 0.1 hr⁻¹ to 0.2 hr⁻¹, the amount ofammonium chloride (AC), or its equivalent, had to be about doubled,e.g., from 0.71 to 1.42, in order to ensure substantially completefermentation of the glucose substrate. By comparison, when the amount ofassimilable nitrogen as ammonium chloride was maintained at 1.07 g/Lwhile the dilution rate was increased 0.15 hr⁻¹ to 0.2 hr⁻¹, the biomassconcentration in the fermenter remained constant at 2.00 g/L, but thefermentation was incomplete as evidenced by the appearance ofunfermented glucose in the fermenter effluent. The glucose concentrationin the effluent (S_(o)) increased from a level of less than 0.5% (w/v)to a level of about 3.0% (w/v) when the concentration of assimilablenitrogen was not adequately controlled.

Nitrogen limitation according to this invention does not appreciablyaffect the efficiency of conversion of substrate to ethanol, expressedas the amount of ethanol produced per amount of substrate utilized,rather than the amount of ethanol produced per the amount of substrateadded to the fermenter. In the fermentations reported in Table 3 thatwere carried out according to this invention, the observed ethanolconcentration was about 52 g/L. This corresponded to a product yield ofabout 93% of the theoretical maximum yield.

Under conditions of excess-nitrogen, the specific rate of glucose uptakeand the specific rate of ethanol production increases with increasingdilution rates. In the case of nutrient-limited fermentation, thebiomass expresses maximal rate of sugar uptake and maximal rate ofethanol production. The rates are higher in all cases withnutrient-limitation than with nutrient-excess. The data in Table 3 showthat a more pronounced effect on the specific rate of glucose uptake andthe specific rate of product formation can be obtained at lower dilutionrates by carrying out the fermentation under nitrogen-limitingconditions according to this invention.

Further work has shown that the advantages to be obtained by the processof this invention are lessened somewhat when compared to a similarprocess conducted with a nutrient medium containing the nitrogen inexcess, at dilution rates below about 0.1 hr⁻¹. The advantages obtainedby the process of this invention are maximized at a dilution rate ofabout 0.1 hr⁻¹.

Data demonstrating that there is typically a maximum imposed on both theconcentration of glucose in the feed stream (S_(r)) to the fermenter andthe dilution rate (D) when complete conversion (95% or more) of sugar toethanol is desired at relatively high ethanol concentrations can befound in Table 4.

                  TABLE 4                                                         ______________________________________                                        Amount of Assimilable Nitrogen as Either Yeast                                Extract (Difco) Ammonium Chloride or Ammonium Sulphate                        Require to Achieve Maximal Rate of Sugar Utilization                          and Ethanol Production by Z. mobilis ATCC 29191 at                            High Product Concentration                                                    ______________________________________                                        D = 0.01 hr.sup.-1                                                            Excess Nitrogen                                                               S.sub.r   Eth    X                                                            g/L       g/L    g/L          q.sub.s                                                                            q.sub.p                                    ______________________________________                                        140*      65     2.5          5.5  2.6                                        ______________________________________                                        Nitrogen Limitation                                                           S.sub.r  Eth  X                  YE    AC    AS                               g/L      g/L  g/L     q.sub.s                                                                            q.sub.p                                                                             g/L   g/L   g/L                              ______________________________________                                        140*     65   1.9     7.1  3.3   4.3   1.00  1.26                             ______________________________________                                         Units: q.sub.s = g glu/g cellhr.sup.-1 ; q.sub.p = g product/g                cellhr.sup.-1.                                                                *NOTE: S.sub.o = 5 g/L.                                                  

When the concentration of glucose in the feed stream was elevated to 140g/L, the maximum dilution rate that could be maintained for completefermentation was 0.1 hr⁻¹. The ethanol concentration in the fermentationmedium was 65 g/L for this glucose level. Table 4 compares the resultsfor nitrogen-limited fermentation according to this invention withfermentation carried out under conditions of nitrogen-excess. The datashow that even with a high concentration of sugar in the feed stream anda high ethanol concentration in the fermentation medium, this inventionmakes it possible to operate at a reduced biomass level, an increasedrate of substrate uptake and an increased rate of product formation.

Potassium-Limited Fermentation

This invention can also be carried out by potassium-limited fermentationof Zymomonas in continuous culture in a manner analogous to thenitrogen-limited fermentation previously described and with comparableresults. The range of tolerable amounts of potassium in the nutrientmedium is rather narrow. For this reason, and for the additional reasonthat the amount of the limiting nutrient must be known with substantialprecision, potassium-limited fermentation is conducted in a definedsalts medium. An example of a suitable medium is described hereinafterwith reference to Table 7.

The amount of potassium, as potassium chloride, required to achievemaximum rate of sugar utilization and ethanol production by Z. mobilisstrain ATCC 29191 in continuous culture in a chemostat at fixed dilutionrate of 0.15 hr⁻¹ was determined as a function of feed sugarconcentration. The results are reported in Table 5.

                  TABLE 5                                                         ______________________________________                                        Amount of Potassium (as Potassium Chloride) Required to                       Achieve Maximal Rate of Sugar Utilization and                                 Ethanol Production by Z. mobilis ATCC 29191 in                                Continuous Culture at Fixed Dilution Rate (0.15 hr.sup.-1)                    as a Function of Feed Sugar Concentration                                     ______________________________________                                        D = 0.15 hr.sup.-1                                                            Excess Potassium                                                              S.sub.r   Eth    X                                                            g/L       g/L    g/L          q.sub.s                                                                            q.sub.p                                    ______________________________________                                        20        9.6    0.58         5.2  2.5                                        60        28     1.73         5.2  2.5                                        110       52     3.17         5.2  2.5                                        ______________________________________                                        Potassium Limitation                                                          S.sub.r                                                                            Eth        X                     KCl                                     g/L  g/L        g/L    q.sub.s   q.sub.p                                                                            g/L                                     ______________________________________                                        20   9.6         0.40  7.5       3.6  0.024                                   60   28         1.2    7.5       3.6  0.071                                   110  52         2.2    7.5       3.6  0.129                                   ______________________________________                                         Units: q.sub.s = g glu/g cellhr.sup.-1 ; q.sub.p = g eth/g cellhr.sup.-1.

As shown in Table 5 and as in the nitrogen-limited fermentation,potassium-limited fermentation can be carried out at lower biomassconcentration than under conditions of potassium-excess at a givenglucose concentration. Also, the fermenter can be operated at a higherq_(p) under potassium-limitation than under potassium-excess at a givenglucose concentration. As glucose in the feed is increased, biomass inthe fermenter also increases, but the biomass level is less underpotassium-limitation than under potassium-excess. As withnitrogen-limited fermentation, the amount of potassium required forpotassium-limited fermentation is proportional to the concentration ofglucose in the feed stream; the amount of potassium as KC1 must beincreased as glucose concentration is increased to maintain q_(p). Onceagain, the nutrient limitation does not appreciably affect product yieldwhen compared with a similar fermentation carried out undernutrient-excess.

When this invention is carried out with potassium-limited fermentationand when it is necessary to control the pH of the fermentation medium, apH regulating agent or buffering agent other than potassium hydroxide orother potassium-containing compound should be employed, otherwise it ispractically impossible to control the amount of potassium in thefermentation medium. The use of sodium hydroxide as the pH regulatingagent in a potassium-limited fermentation has been found to beadvantageous. Sodium apparently acts as a potassium antagonist, and theresulting elevated level of sodium after the addition of sodiumhydroxide to the fermenter potentiates the effect ofpotassium-limitation on the specific activity of the biomass. Abuffering agent, such as NaH₂ PO₄, can also be employed.

Phosphorus-Limited Fermentation

This invention can be carried out by phosphorus-limited fermentation ofZymomonas in continuous culture in a manner analogous to thenitrogen-limited fermentation previously described and with comparableresults. As with potassium-limited fermentation, the range of tolerableamounts of phosphorus in the nutrient medium is rather narrow. For thisreason, and for the additional reason that the amount of the limitingelement must be known with substantial precision, phosphorus-limitedfermentation is conducted in a defined salts medium. Once again, anexample of a suitable medium is described hereinafter with reference toTable 7.

The amount of assimilable phosphorus as potassium dihydrogen phosphaterequired to achieve maximal rate of sugar utilization and ethanolproduction by Zymomonas mobilis strain ATCC 29191 in continuous culturein a chemostat at fixed dilution rate of 0.15 hr⁻¹ was determined as afunction of feed sugar concentration. The amount of potassium dihydrogenphosphate employed and the results obtained are reported in Table 6.

                  TABLE 6                                                         ______________________________________                                        Amount of Assimilable Phosphorus, as KH.sub.2 PO.sub.4,                       Required to Achieve Maximal Rate of Sugar                                     Utilization and Ethanol Production by                                         Z. mobilis ATCC 29191 in Continuous Culture                                   at Fixed Dilution Rate (0.15 hr.sup.-1)                                       as a Function of Feed Sugar Concentration                                     ______________________________________                                        D = 0.15 hr.sup.-1                                                            Excess Phosphate                                                              S.sub.r   Eth    X                                                            g/L       g/L    g/L          q.sub.s                                                                            q.sub.p                                    ______________________________________                                        20        9.6    0.58         5.2  2.5                                        60        28     1.73         5.2  2.5                                        110       52     3.17         5.2  2.5                                        ______________________________________                                        Phosphate Limitation                                                          S.sub.r                                                                            Eth       X                    KH.sub.2 PO.sub.4                         g/L  g/L       g/L    q.sub.s  q.sub.p                                                                            g/L                                       ______________________________________                                        20   9.6       0.42   7.2      3.4  0.04                                      60   28        1.25   7.2      3.4  0.13                                      110  52        2.29   7.2      3.4  0.23                                      ______________________________________                                         Units: q.sub.s = g glu/g cellhr.sup.-1 ; q.sub.p = g eth/g cellhr.sup.-1.

The data in Table 6 show that the results obtained withphosphorus-limited fermentation are analogous to the results obtainedwith nitrogen-limited and potassium-limited fermentations.Phosphorus-limited fermentation can be carried out at lower biomassconcentration, higher specific rate of glucose uptake, higher specificrate of product formation and comparable product yield as compared tofermentation carried out under conditions of phosphorus-excess. The datashow that the amount of phosphorus required for phosphorus-limitedfermentation is proportional to the concentration of glucose in the feedstream.

When this invention is carried out with phosphorus-limited fermentationand when it is necessary to control the pH of the fermentation medium, apH regulating agent or buffering agent other than aphosphorus-containing compound should be employed. The additional sourceof phosphorus from a phosphorus-containing pH regulating agent mayprevent the degree of control of nutrient-limitation required by theinvention. For phosphorus-limited fermentation KH₂ PO₄ can be employedas a buffer and NaOH titrant diluted to 0.5N in order to avoid largechanges in pH during automatic titration.

Formulating the Nutrient Medium

The identity of the chemical constituents in the nutrient medium and theamount of each constituent should be sufficient to meet the elementalrequirements for cell mass and ethanol production and should supplyappropriate energy for synthesis and maintenance. The nutrient mediumshould contain carbon, nitrogen, potassium, phosphorus, sulfur,magnesium, calcium and iron in required amounts. The chemicalconstituents should also meet specific nutrient requirements includingvitamins and trace minerals.

As the assimilable source of nitrogen, various kinds of inorganic ororganic salts or compounds can be included in the nutrient medium. Forexample, ammonium salts, such as ammonium chloride or ammonium sulfate,or natural substances containing nitrogen, such as yeast extract,peptone, casein hydrolysate or corn steep liquor, or amino acids, suchas glutamic acid, can be employed. These substances can be employedeither singly or in combination of two or more.

Examples of inorganic compounds that can be included in the culturemedium are magnesium sulfate, potassium monohydrogen phosphate,potassium dihydrogen phosphate, sodium chloride, magnesium sulfate,calcium chloride, iron chloride, magnesium chloride, zinc sulfate,cobalt chloride, copper chloride, borates and molybdates.

Organic compounds that may be desirable in the fermentation include, forexample, vitamins, such as biotin, calcium pantothenate, and the like,or organic acids, such as citric acid, or amino acids, such as glutamicacid. It has been found, however, that biotin is not required in thegrowth medium.

Fermentation acids that are non-toxic to the microorganism can beincluded in the nutrient medium and fermentation broth. For example, ananti-foaming agent in a minor amount has been found to be advantageous.

Examples of nutrient media that have been found suitable for use in thisinvention are described in Table 7.

                  TABLE 7                                                         ______________________________________                                        Chemical Composition of Growth Media for                                      Continuous Culture of Z. mobilis                                                         SEMI-SYNTHETIC DEFINED                                                        MEDIUM         SALTS MEDIUM                                        INGREDIENT (g/L)          (g/L)                                               ______________________________________                                        D-Glucose  100            100                                                 (anhydrous)                                                                   approx.                                                                       Yeast Extract                                                                             5             --                                                  (Difco)                                                                       NH.sub.4 Cl                                                                              2.4            2.4                                                 KH.sub.2 PO.sub.4                                                                        3.48           3.48                                                MgSO.sub.4.7H.sub.2 O                                                                    1.0            1.0                                                 FeSO.sub.4.7H.sub.2 O                                                                    0.01           0.01                                                Citric Acid                                                                              0.21           0.21                                                Vitamins                                                                      Ca-pantothenate                                                                           0.001          0.001                                              Biotin      0.001          0.001                                              Antifoam                                                                      as required                                                                   ______________________________________                                    

The semi-synthetic medium is suitable for use in the nitrogen-limitedfermentation according to this invention. It will be understood that thecomposition of this medium will depend on the technical quality of thenitrogen source. For example, yeast extract from a batch from onecommercial source may exhibit a different potency with respect to thecontent of assimilable nitrogen than a yeast extract from a differentbatch or from another commercial source. Also, the amount of thenitrogen source required in the medium will depend on the degree ofhydration; anhydrous chemicals are preferred and were employed in theExamples and in the fermentations reported in the Tables.

The defined salts medium is suitable for use in carrying out thepotassium-limited and phosphorus-limited fermentations. Only inorganicsources of nitrogen, such as ammonium salts, are employed in definedsalts media. In the experiments reported in the foregoing Tables andExamples, the defined salts medium was used in the potassium- andphosphorus-limited fermentations, with the following exceptions.

Ordinarily, the phosphorus in the nutrient medium is supplied as a salthaving an anion that is substantially non-toxic to the microorganism andthat does not substantially inhibit normal metabolic processes. While apotassium salt, such as potassium dihydrogen phosphate, is typicallyemployed for nitrogen-limited and phosphorus-limited fermentations, asodium salt is preferably substituted for the potassium salt inpotassium-limited fermentation. For example, sodium dihydrogen phosphateinstead of potassium dihydrogen phosphate can be utilized. Since theamount of available potassium must be precisely known underpotassium-limited conditions, this substitution makes it easier tocontrol the relative proportions of nutrients.

The potassium in the nutrient medium for potassium-limited fermentationis supplied by a salt having an anion that is substantially non-toxic tothe microorganism and that does not substantially inhibit normalmetabolic processes. The source of potassium is preferably potassiumchloride, although similar water-soluble, inorganic salts can beemployed.

The amount of limiting nutrient in the nutrient medium mainly depends ontwo factors: The concentration of substrate in the feed stream to thefermenter and the dilution rate. As the substrate concentration at aconstant dilution rate is increased, the amount of limiting nutrient isincreased. Similarly, at a constant substrate concentration, the amountof limiting nutrient is increased as the dilution rate increases. Theserelationships apply to nitrogen-limited, potassium-limited andphosphorus-limited fermentations, since the fermentations are analogousto each other. The concentrations of inorganic salts other than the N-,K- and P-containing salts are relatively invariant with the formulationsshown in Table 7.

The nutrient media in Table 7 can also be employed in a fermentationcarried out under conditions of nutrient excess. For example, anitrogen-excess medium based on yeast extract (Difco) and ammonium ioncan contain about 5 to about 10 g/L of the yeast extract and about 15 toabout 34 mM, preferably about 30 mM, ammonium ion. Molar values, aregiven because the weight depends on the particular ammonium salt chosen.For ammonium chloride the corresponding concentrations would be about0.8 to about 2.4 g/L, preferably about 1.6 g/L. The nutrient medium usedin the fermentations carried out under conditions of nitrogen-excess andreported in Tables 2, 3 and 4 was the semi-synthetic medium of Table 7containing 5 g/L yeast extract (Difco) and 1.6 g/L NH₄ Cl.

The amount of limiting nutrient, namely nitrogen, potassium orphosphorus, expressed as the concentration in the growth medium beingfed to the fermenter, required to achieve a condition of growthlimitation can be determined from Equations (1) and (2) and a knowledgeof values for the growth yield with respect to the particular limitingnutrient and the maximum specific rate of substrate utilization (q_(s)^(max)). While these values may be strain specific, they can beexperimentally determined and examples are given below.

The equations used to calculate the amount of limiting nutrient requiredto achieve a condition of nutrient deficiency or limitation are:

    N*=X */Y.sub.n                                             (1)

where

N*=the amount of source of nutrient, g;

X*=the dry mass of cells (dry wt biomass), g; and

Y_(n) =growth yield coefficient for a specified limiting nutrient, n; gdry biomass/g atom nutrient source (see Table 9).

The value for X* in Equation (1) is determined by Equation (2):

    X*=S.sub.r (D)/q.sub.s.sup.max                             (2)

where

S_(r) =the concentration of substrate in the feed stream to thefermenter, g/L;

D=the dilution rate, hr⁻¹ ; and

q_(s) ^(max) =the maximum observed specific rate of substrate uptake forthe strain of Zymomonas being used in the continuous fermentation, gglu/g biomass-hr⁻¹ ; (see Table 10); and wherein S_(r) ≦140 g/L andD≧0.1 hr⁻¹.

Experimentally determined values of various growth yield coefficients(Y_(n)) for Z. mobilis strain ATCC 29191 with respect to different solesources of assimilable nitrogen are given in Table 8.

                  TABLE 8                                                         ______________________________________                                        Observed Values of Growth Yields                                              (Z. mobilis ATCC 29191)                                                       Nitrogen Source  Growth Yield Y.sub.n                                         ______________________________________                                        Yeast Extract (Difco)                                                                          0.45 g dry biomass/g YE                                      Ammonium Chloride                                                                              1.87 g dry biomass/g NH.sub.4 Cl                             Ammonium Sulphate                                                                              1.52 g dry biomass/g                                                          (NH.sub.4).sub.2 SO.sub.4                                    ______________________________________                                    

Growth yield coefficients (Y_(n)) with respect to nitrogen, phosphorusand potassium were calculated from steady-state biomass concentrationsin respectively limited chemostat cultures at a fixed dilution rate of0.15 hr⁻¹, a constant temperature of 30° C. and a pH of 5.5. In eachcase the entering glucose concentration was approximately 100 g/L. Theresults are summarized in Table 9. The value of growth yield withrespect to potassium is influenced by the [Na⁺ ] such that Y_(K)decreases with increasing concentration of Na⁺ in the culture medium.The titrant used to maintain pH was NaOH. Observed values for differentgrowth yields for Z. mobilis are in good agreement with general valuescited in the literature with respect to the elemental composition (%w/w) of bacteria.

                  TABLE 9                                                         ______________________________________                                        Calculated Values of Growth Yields                                            (Z. mobilis ATCC 29191)                                                       Type of                    Composition                                        Limiting     Y.sub.n Growth Yield                                                                        of Biomass                                         Nutrient     (g biomass/g atom)                                                                          % w/w                                              ______________________________________                                        Nitrogen (N) 7.1           14                                                 Phosphorus (P.sub.i)                                                                       44            2.3                                                Potassium (K.sup.+)                                                                        33            3.0                                                ______________________________________                                    

The values given in Table 9 can be substituted in Equation (1). It willbe understood that these values may vary with the fermentation systemand operating techniques and should be confirmed by experimentation inthe system under study or in question.

It has been suggested in the literature that the values for q_(s) ^(max)and q_(p) ^(max) are stain specific traits in Z. mobilis. In any event,q_(s) ^(max) and q_(p) ^(max) for the strain of interest can best bedetermined by means of non-carbon limitation under steady-stateconditions in continuous culture in a chemostat. The value for q_(s)^(max) may vary depending on the nature of the limiting nutrient and theparticular strain of Z. mobilis chosen, but q_(s) ^(max) is generallyabout 7 to 10 g glucose/g cell-hr⁻¹.

Experminetally determined values of q_(s) ^(max) for nutrient-limitedfermentations by the strain ATCC 29191 are given in Table 10.

                  TABLE 10                                                        ______________________________________                                        Observed Average Values of Maximum Specific Rate of                           Glucose Uptake for Nutrient-Limited                                           Fermentation by Strain ATCC 29191 in                                          Continuous Culture in a Chemostat                                             Type of                                                                       Limiting   Chemical    q.sub.s max                                                                              Y.sub.X /n                                  Nutrient   Identity of (g glucose/g                                                                             (g/glu/g                                    n          Nutrient    biomass-hr.sup.-1                                                                        of n                                        ______________________________________                                        Nitrogen   NH.sub.4 Cl 8.3        1.87                                                   (NH.sub.4).sub.2 SO.sub.4                                                                 8.3        1.52                                                   Yeast Extract                                                                 (Difco)     8.3        0.45                                        Potassium  KCl         7.5        17                                          Phosphate  KH.sub.2 PO.sub.4                                                                         7.2        10                                          ______________________________________                                    

The values in Table 10 can be substituted in Equation (2), once againsubject to confirmation in the system under study.

The observed q_(s) ^(max) for potassium-limited Z. mobilis strain ATCC29191 has been found to be 7.5 g glucose/g biomass-hr⁻¹, and the valuefor the growth yield (Y_(K)) with respect to KCl has been found to be 17g biomass/g KC1. The observed value for Y_(K) is 33 g biomass/g K⁺,which is equivalent to saying that the biomass is 3% w/w potassium.Equations (1) and (2) can also be used to predict the amount ofpotassium (e.g., as KC1) required to achieve a condition of potassiumlimitation in continuous culture at various values for the concentrationof glucose in the feed stream (S_(r)) and dilution rate (D).

The observed average value for q_(s) ^(max) for a phosphorus-limitedculture of Z. mobilis is 7.2 g glu/g biomas-hr⁻¹ and the growth yieldwith respect to phosphorus (as potassium dihydrogen phosphate) is 10 gbiomass/g KH₂ PO₄. The observed Y_(p) is 44 g biomass/g P, which isequivalent to biomass being 2.3% w/w with respect to its phosphoruscontent. These values can be substituted appropriately into Equations(1) and (2) in order to predict the amount of phosphorus required toachieve a condition of phosphorus-limitation at various values of S_(r)and dilution rate.

The values in Tables 8 and 9 were determined at various values for S_(r)and D. As mentioned previously, because Z. mobilis is sensitive toethanol at concentrations in excess of about 5% (w/v), there is an upperlimit to the practical value of S_(r), namely about 150 g fermentablesugar/L.

The following Examples illustrate working embodiments of this invention.

Example 1

Continuous ethanol fermentations were preformed in apparatus similar tothat described in the Figure Bench-top chemostat (Model C30, NewBrunswick Scientific Co. N.J.) were used in which the constant workingvolume (V) of 350 ml was established and maintained by means of anattached overflow tube. The culture was agitated by means of a pair ofturbine six-blade impellers operating at 200 RPM. The temperature wascontrolled at 30° C. and the pH was monitored using a combination Ingoldelectrode coupled to a Model pH-40 (New Brunswick Scientific ) pHanalyzer. The addition of titrant (KOH) was controlled automatically bythe pH controller and maintained at 5.5. The vessel was not sparged withgas of any kind except during start-up when oxygen-free N₂ was used at arate of approximately 0.5 v/v/m.

The chemical composition of the semi-synthetic culture medium isdescribed in Table 7. The concentration of glucose was 20, 60 or 110g/L. Yeast extract obtained from Difco was the sole source ofassimilable nitrogen added to the culture medium (i.e., ammoniumchloride was not added). In order to achieve a condition ofnitrogen-excess growth, 5 g/L yeast extract were added to the basalsalts medium (at all concentrations of glucose). For nitrogen-limitationthe amount of yeast extract added to the salts medium depended on theamount of glucose in the medium such that for media containing 20, 60and 110 g glucose/L, yeast extract in amounts of 0.8, 2.4 and 4.4 g,respectively, were added per liter (L).

Polypropylene glycol 2025 was added to the medium as an antifoamingagent at a concentration of 0.1 ml/L. Media were prepared and autoclavedin 13 L pyrex carbuoys. Sterile culture medium was fed to the fermenterat a constant rate (F) by means of a peristaltic pump such that thedilution rate (calculated as F/V) was 0.15/hr⁻¹. The fermenter wasinoculated (15% v/v) with Z. mobilis ATCC 29191, which had been grownovernight in medium of similar composition in a non-agitated flaskincubated at 30° C. Flow to the fermenter was not commenced until theculture was in late-exponential phase of growth. Growth and biomassconcentration were determined as dry weight of culture collected onpreweighed microporous filters (Millipore Corp., 0.45 μm pore size).Sampling the biomass and weighing the dry cells has been found to bemuch more accurate and reliable than turbidity measurement ormeasurements made by indirect methods. Steady-state was presumed to haveoccurred after a minimum of 4 culture turnovers, a turnover beingequivalent in time to the reciprocal of the dilution rate. Glucose wasdetermined using a YSI Glucose Analyzer (Model 27, Yellow SpringsInstrument Co., Ohio). Ethanol was measured by HPLC (HPX-87H Aminex,300×7.8 mm column, from Bio-Rad, Burlington, Ont. Can.). The culture wasroutinely examined for contamination both by microscopic assessment andby plating on selective diagnostic agar media.

The specific rate of glucose uptake, q_(s), (g glucose consumed/gbiomass-hr⁻¹), was calculated as follows: ##EQU1## where S_(r) and S_(o)represent the concentration of fermentable sugar in the feed reservoirand fermenter effluent, respectively;

D=the dilution rate (hr⁻¹); and

X=the dry weight culture biomass concentration (g/L).

Similarly, the specific rate of ethanol formation, q_(p), (g ethanol/gbiomass-hr⁻¹) was calculated as follows: ##EQU2## where [P] representsthe steady-state ethanol concentration.

The results of this experiment are summarized in Table 2.

EXAMPLE 2

The same procedure was followed as in EXAMPLE 1 except the sole sourceof nitrogen was ammonium chloride (no yeast was added to the medium andas such it is referred to as a defined salts medium). Table 2 shows theamount of ammonium chloride (NH₄ Cl) used at different values for S_(r)with respect to glucose, these being 0.19, 0.59 and 1.10 g NH₄ Cl/L forS_(r) glucose values of 20, 60 and 110 g/L, respectively.

EXAMPLE 3

The same procedure was followed as in EXAMPLE 1 except that the solesource of assimilable nitrogen was ammonium sulphate (AS). The amountsadded and the results obtained are shown in Table 2.

EXAMPLE 4

Experiments were preformed with Z. mobilis strain ATCC 31821. Theresults were substantially the same as those observed with strain ATCC29191.

EXAMPLE 5

Experiments were performed with Z. mobilis strain ATCC 10988. Theresults were substantially the same as those observed with strain ATCC29191.

EXAMPLE 6

Experiments were performed to show the amount of assimilable nitrogenrequired to achieve nitrogen-limitation as a function of dilution rate.

The results are summarized in Table 3. S_(r) was constant at 110 g/L andthe dilution rate was set at 0.1, 0.15 and 0.2 hr⁻¹. The sole sources ofnitrogen were yeast extract (Difco), ammonium chloride and ammoniumsulphate. Although the results shown in Table 3 were obtained withstrain ATCC 29191, substantially similar results were observed with bothATCC 31821 and ATCC 10988.

EXAMPLE 7

Table 4 summarizes the results of an experiment with ATCC 29191 toillustrate the effect of end-product (ethanol) inhibition on the generalformula for predicting fermentation performance under conditions ofnitrogen excess and nitrogen limitation. Even when the continuousfermenter was operated near its upper limit with respect to ethanolconcentration, the specific activities (q_(s) and q_(p)) of the culturewere improved by imposing the condition of nitrogen limitation.

EXAMPLE 8

Experiments were performed to show the amount of potassium required toachieve potassium-limitation as a function of glucose concentration at aconstant dilution rate for ATCC 29191. D was constant at 0.15 hr⁻¹, thepH was controlled with NaOH at 5.5 and S_(r) was set at 20, 60 and 110g/L. The sole source of potassium was KCl. The concentration of KCl andresults obtained are summarized in Table 5.

EXAMPLE 9

Experiments were performed to show the amount of assimilable phosphorus,as KH₂ PO₄, required at various sugar concentrations to achieve maximalrate of sugar utilization and ethanol production by Z. mobilis strainATCC 29191, in continuous culture in a chemostat at a fixed dilutionrate of 0.15 hr⁻¹. A pH of 5.5 was maintained with KOH. Theconcentrations of KH₂ PO₄ and the results obtained are shown in Table 6.

EXAMPLE 10

Continuous ethanol fermentation was performed in apparatus similar tothat shown in FIG. 1. A bench-top chemostat (Bioflo Model C30, NewBrunswick Scientific Co., Edison, N.J.) was used in which a constantworking volume (V) of 350 ml was established and maintained by means ofan attached overflow tube. The culture was agitated by means of a pairof turbine, six-blade impellers operating at 200 RPM. The temperaturewas controlled at 30° C.

The pH was monitored using a combination Ingold electrode coupled to aModel pH-40 (New Brunswick Scientific) pH analyzer. The pH wasautomatically maintained at pH values within the range of 4.0 to 7.5 bythe addition of titrant (3N KOH) by the pH analyzer. The vessel wassparged with oxygen-free N₂ gas at a rate of approximately 50-100cc/min.

The chemical composition of the semi-synthetic culture medium used isgiven in TABLE 7.

Yeast extract (0.3% by weight) obtained from Difco was added to theculture medium as a source of assimilable nitrogen. Also, ammoniumchloride (30 mM) was added as an additional source of nitrogen. Thefermentation was thus carried out under a condition of nitrogen-excess.Polypropylene glycol 2025 was added to the medium as an antifoamingagent at a concentration of 0.1 ml/L.

Media were prepared and autoclaved in 13 L pyrex carbuoys. Sterileculture medium was fed to the fermenter at a constant rate (F) by meansof a peristaltic pump such that the dilution rate (calculated as F/V)was 0.15/hr⁻¹. The fermenter was inoculated (15% v/v) with Z. mobilisATCC 29191, which has been grown overnight in medium of similarcomposition in a non-agitated flask incubated at 30° C.

Flow to the fermenter was not commenced until the culture was inlate-exponential phase of growth. The concentration of glucose fed tothe fermenter was 50 g/L.

Growth and biomass concentration were determined as dry weight ofculture collected on preweighed microporous filters (Millipore Corp.,0.45 m pore size). Sampling the biomass and weighing the dry cells hasbeen found to be much more accurate and reliable than turbiditymeasurement or measurements made by indirect methods. Steady state waspresumed to have occurred after a minimum of 4 culture turnovers, aturnover being equivalent in time to the reciprocal of the dilutionrate.

Glucose was determined using a YSI Glucose Analyzer (Model 27, YellowSprings Instrument Co., Ohio). Ethanol was measured by HPLC (HPX-87HAminex, 300×7.8 mm column, from Bio-Rad, Burlington, Ont. Can.). Theculture was routinely examined for contamination both by microscopicassessment and by plating on selective diagnostic agar media.

The specific rate of glucose uptake, q_(s), and the specific rate ofethanol formation, q_(p), were calculated as described in Example 1. Theresults are summarized in Table 10a.

                  TABLE 10a                                                       ______________________________________                                        The Effect Of pH On Fermentation Kinetics And Yield                           Coefficients Of Z. mobilis ATCC 29191 In Continuous                           Culture (5% glu, 30° C., D = 0.15 hr.sup.-1)                                   Qs       Qp         Yx/s                                              pH      (g/g/hr) (g/g/hr)   (g/g) [P] (g/L)                                   ______________________________________                                        4.0     8.35     3.36       0.0198                                                                              21.8                                        4.5     7.9      3.0        0.022 21.8                                        5.0     6.45     2.65       0.0245                                                                              21.8                                        5.5     5.2      2.2        0.0316                                                                              21.6                                        6.0     4.75     1.9        0.033 21.2                                        6.5     5.0      2.12       0.031 21.4                                         7.0*   --       --         --    16.0                                         7.5*   --       --         --    --                                          ______________________________________                                         *Flocculation of biomass.                                                

The results reported in Table 10a are plotted in FIGS. 3, 4, and 5 andthe Figures are described above. The abbreviation "e-2" along theordinate of FIG. 4 is used herein to mean "x10⁻² ".

The results of experiments designed to examine the effect of pH on theperformance of Zymomonas under steady-state growth conditions (D=0.15hr⁻¹, T=30° C., the complex medium contained 5% glucose with 30 mM (NH₄Cl) are summarized in TABLE 10a. There was complete utilization of theglucose over the pH range from 4.0 to 6.5, but the culture washed-out ofthe chemostat when the pH was decreased below 3.8. Since cultureresponse to step changes in pH are often slow, a minimum of 6 turnovers(volume changes) was permitted before assuming steady-state growth. Asevidenced by the value of the steady-state growth yield (Y_(x/s)),energy conservation appears to be maximal at pH 6.0, but as the pHdecreases from 6.0 to 4.0 there is an apparent increasing degree ofenergetic uncoupling. In a separate series of growth experiments(results not shown), the effect of pH on q_(s) was assessed as afunction of the dilution rate (growth rate), and from an extrapolationof the linear relationship between q_(s) and D, it was concluded thatthe pH affects m_(e) and not Y_(g) ^(max) (y_(g) ^(max) =maximum molargrowth yield with respect to carbon). A similar conclusion was reachedin connection with the effect of temperature on Zymomonas where thevalue of m_(e) increased from 0.5 to 2.5 g glu/g cell/hr with increasingtemperature from 30° to 35° C. whereas Y_(g) ^(max) was unaffected.Biotechnol. Bioeng., 25, 1655 (1983).

Effect of Temperature on Nutrient-Limited Fermentation

Microbial growth and product formation are the result of a complexseries of biochemical reactions that are temperature dependent.Zymomonas strains have a broad range of temperatures within whichmetabolic processes will occur and an optimum temperature range withinwhich the rate of product formation increases. Above the optimumtemperature, the rate of product formation rapidly declines, due in partto an increasing cell death rate and reduced cell growth rate. Lowerbiomass level translates to incomplete fermentation in a continuoussystem unless the dilution rate is adjusted appropriately downwardly.This is because, at relatively high substrate concentrations in thefermenter feed, the specific rate of substrate uptake will increase withincreasing temperature, but the reduced biomass decreases the capacityof the fermenter to process the same substrate load. Heretofore, theoptimum temperature was not exceeded when the object was to obtainmaximum conversion of substrate to product in the least possible time.

The effect of temperature on the maximum specific growth rate (u_(max))of Z. mobilis strain ATCC 29191 in batch culture has been reported inthe literature and is shown in Table 11. The culture medium contained 2%(w/v) glucose.

                  TABLE 11                                                        ______________________________________                                        The Effect of Temperature on the Maximum Specific                             Growth Rate of Z. mobilis ATCC 29191 in                                       Batch Culture (2% glu and pH 5.5)                                             Temperature (°C.)                                                                      .sup.u max (hr.sup.-1)                                        ______________________________________                                        30              0.27                                                          33              0.38                                                          36              0.26                                                          ______________________________________                                    

The data in Table 11 shown that as the temperature in the culture mediumwas increased from 30° C. to 33° C., the maximum specific growth rateincreased, but with a further increase in temperature from 33° C. to 36°C., the maximum specific growth rate declined. Thus, the optimumfermentation temperature for this system was about 33° C. Cell growthwas inhibited above this temperature.

While increasing temperature affects cell growth as shown in Table 11,it has now been found that increasing temperature also affects substrateconversion and product formation. Table 12 shows the effect ofincreasing temperature on performance of a carbon-limited continuousfermentation by Z. mobilis strain ATCC 29191 in a chemostat at aconstant dilution rate of 0.19 hr⁻¹. The substrate was fed to thechemostat as an aqueous solution containing 2% (w/v) glucose.

                  TABLE 12                                                        ______________________________________                                        Effect of Increasing Temperature on Performance of                            Carbon-Limited Continuous Fermentation by                                     Z. mobilis ATCC 29191 at Constant Dilution Rate                               (D = 0.19 hr.sup.-1 ; 2% glu; pH 5.5)                                                   30° C.                                                                           33° C.                                                                         36° C.                                     ______________________________________                                        S.sub.r (g/L)                                                                             20.7        20.7    20.7                                          S.sub.o (g/L)                                                                             --          0.1     0.5                                           X (g/L)     0.65        0.55    0.45                                          q.sub.s (g/g-hr.sup.-1)                                                                   5.9         7.25    8.8                                           q.sub.p (g/g-hr.sup.-1)                                                                   2.80        3.41    4.14                                          Y.sub.p /s(g/g)                                                                           0.47        0.47    0.47                                          ______________________________________                                    

In the continuous fermentations reported in Table 12, the glucoseconcentration in the feed to the fermenter was maintained constant at20.7 g/L. When the temperature in the fermentation medium was increasedfrom 30° C., the biomass concentration (X) in the fermenter decreased,but this decrease was compensated for by an increase in the specificrate of glucose uptake (q_(s)). It will be observed, however, thatglucose began to appear in the fermenter effluent (i.e., S_(o) =0.1 g/L)at 33° C.

When the temperature in the fermentation medium was further increasedfrom 33° C. to 36° C., once again the biomass concentration in thefermenter declined, this time from 0.55 g/L to 0.45 g/L, but the declinewas offset by a further increase in the specific rate of glucose uptake(q_(s)). The conversion of glucose to ethanol was incomplete and theconcentration of glucose in the effluent (S_(o)) increased to 0.5 g/L.While the yield coefficient (Y_(p/s)) remained constant at 0.47 g/g, thepresence of uncoverted glucose in the effluent was unacceptable.

The effect of increasing temperature on the perfomance of K⁺ -limitedcontinuous fermentation by Z. mobilis strain ATCC 29191 in a chemostatat a constant dilution rate of 0.15 hr⁻¹ is shown in Table 13. Potassiumwas supplied as KCl at a concentration of 0.13 g/L.

                  TABLE 13                                                        ______________________________________                                        Effect of Increasing Temperature on Performance of                            K.sup.+ -limited Continuous Fermentation by                                   Z. mobilis ATCC 29191 at Consant Dilution Rate                                (D = 0.15 hr.sup.-1)                                                                         30° C.                                                                       35° C.                                            ______________________________________                                        S.sub.r g/L      110     110                                                  [KCl] g/L        0.13    0.13                                                 X (g/L)          2.2     1.48                                                 q.sub.s (g/g-hr.sup.-1)                                                                        7.5     8.9                                                  Y.sub.KCl (g/g)  17      11.5                                                 S.sub.o (g/L)    --      22                                                   ______________________________________                                    

The data in Table 13 show that increasing the temperature in thefermentation medium from 30° C. to 35° C. at a constant glucoseconcentration in the feed stream of 110 g/L resulted in a decrease inthe biomass concentration and an increase in the specific rate ofglucose uptake (q_(s)). In this case, however, the yield coeffient(Y_(n) where n=KCl) declined from 17 g/g KCl to 11.5 g/g KCl and theglucose concentration in the effluent increasd from 0 g/L to 22 g/L.These changes in system performance would adversely affect processeconomics in a commerical operation.

Table 14 shows the effect of increasing temperature on performance of aK⁺ -limited continuous fermentation by strain ATCC 29191 in a chemostatat varying dilution rates. The substrate was fed to the fermenter as anaqueous solution containing 2% (w/v) glucose and at a fixed flow rate.Potassium was supplied as KCl at a concentration of 0.1 g/L.

                  TABLE 14                                                        ______________________________________                                        The Effect of Temperature on Kinetics of                                      K.sup.+ -limited Z. mobilis Strain ATCC 29191                                                                            q.sub.p                            Temp. S.sub.r S.sub.o  D     X     q.sub.s (g/g-                              (°C.)                                                                        (g/L)   (g/L)    (hr.sup.-1)                                                                         (g/L) (g/g-hr.sup.-1)                                                                       hr.sup.-1)                         ______________________________________                                        30.0  100     18.3     0.155 1.70  7.40    3.48                               32.8  100     10.6     0.157 1.68  8.20    3.85                               35.0  100     34.8     0.160 1.15  8.90    4.0                                ______________________________________                                    

Increasing the temperature by 2°-3° C. resulted in more conversion ofglucose to ethanol, i.e. from approximately 82% to 90%, as judged by thedecrease in effluent glucose (S_(o)). However, further increase intemperature caused more glucose to appear in the effluent and theconversion fell to 65%. At 35° C. the morphology of the culture changeddramatically becoming very filamentous. The result of operation at 35°C. was a reduced cell density, and even though the q_(p) was higher, thereduced biomass could not handle the sugar load.

The effect of increasing temperature on the performance of a K⁺ -limitedcontinuous fermentation by Z. mobilis according to this invention isshown in Table 15. The fermentation was carried out by Z. mobilis strainATCC 29191 in a chemostat at a constant dilution rate of 0.15 hr⁻¹.Potassium was supplied as KCl at concentrations indicated.

                  TABLE 15                                                        ______________________________________                                        Effect of Increasing Temperature on Performance                               of K.sup.+ -limited Continuous Fermentation by                                Z. mobilis Strain ATCC 29191 at Constant Dilution Rate                        (D = 0.15 hr.sup.-1)                                                                    30° C.                                                                           35° C.                                                                         35° C.                                     ______________________________________                                        S.sub.r g/L 110         110     110                                           [KCl] g/L   0.13        0.13    0.16                                          X (g/L)     2.2         1.48    1.85                                          q.sub.s (g/g-hr.sup.-1)                                                                   7.5         8.9     8.9                                           Y.sub.KCl (g/g)                                                                           17          11.5    11.5                                          S.sub.o (g/L)                                                                             --          22      --                                            ______________________________________                                    

The concentration of substrate to the fermenter was the same in allcases, i.e., 110 glucose/L. Comparing column 2 with column 3 in Table15, when the ferrmentation temperature was increased from 30° C. to 35°C., the biomass concentration (X) decreased from 2.2 g/L to 1.48 g/L,while the specific rate of glucose uptake (q_(s)) increased from 7.5 to8.9 g/g-hr⁻¹. The amount of glucose in the effluent (S_(o)) alsoincreased from 0 to 22 g glucose/L, which was commercially unacceptable.

Comparing column 2 with column 4 in Table 15, it is seen that byincreasing the concentration of the limiting nutrient according to thisinvention, i.e. [KCl] in this case, when the temperature was increasedfrom 30° C. to 35° C., the biomass concentration (X) in the fermenterdecreased from 2.2 g/L to 1.85 g/L while the specific rate of glucoseuptake (q_(s)) increased from 7.5 to 8.9 g/g-hr⁻¹. However, there wassubstantially no glucose in the effluent from the fermenter when theconcentration of the limiting nutrient was properly contolled, i.e.,S_(o) =0.

These results demonstrate that this embodiment of the invention makes itpossible to improve the performance of Zymomonas in continuous ethanolfermentation at increased temperatures. The specific rate of substrateuptake can be maintained, and even increased, at fermentationtemperatures of about 33° C. to about 37° C. even though there is alower biomass concentration in the fermenter. These results can beachieved without substantial amounts of substrate in the effluent fromthe fermenter. These results are made possible by carrying outcontinuous ethanol fermentation with Zymomonas strains undernutrient-limited conditions, where the limiting nutrient is nitrogen,potassium or phosphorus. The concentration of the limiting nutrient inthe fermentation medium is increased with increasing temperature anddecreased with decreasing temperature.

The effect of pH on u_(max) was determined during exponential growth ofZ. Mobilis ATCC 29191 in batch cultures conducted in stirred fermentersfitted with pH and temperature (30° C.) control. The complex mediumcontained 0.3% yeast extract, 15 mM NH₄ Cl and 5% glucose and theresults are shown in FIG. 2. Under the conditions specified, the pHrange which produced the fastest growth rate was 5.5 to 6.5 with anapparent optimum at pH 6.0 (FIG. 2).

Comparative steady-state data for yeast and Zymomonas continuousfermentations (based on 10% glucose) is shown in TABLE 16. Themaintenance energy coefficient (m_(e)) was derived by extrapolation asthe y-axis intercept in plots of q_(s) versus the dilution rate (D)according to the relationship derived by Pirt whereby ##EQU3## SeePrinciples of Microbe and Cell Cultivation, Blackwell ScientificPublications (1975), p. 67 and Proc. Royal Soc. Lond., Series B, 165,224 (1965). The slope of this plot yields the reciprocal of the maximumgrowth yield (Y_(x/s) ^(max)) corrected for the maintenance energy. Thecorresponding value for the specific rate of ethanol production is afunction of the product yield (Y_(p/s)) or the efficiency with which thefermentable carbon substrate is converted to ethanol since q_(p) =q_(s)(Y_(p/s)). The product yields associated with yeast and Zymomonasfermentations are 0.43 and 0.47 g EtOH/g glu, respectively.

Ethanol production by yeast has been shown to be almost completely agrowth related process; the specific rate being approximately 35 timesfaster in the case of fast growing yeast compared to non-growing yeast(TABLE 16). Maintenance metabolism by non-growing yeast accounts forsome alcohol production, but the rate is comparatively extremely slow(TABLE 16). Ethanol and CO₂ are by-products of energy metabolism andthey are produced in response to the energy demands of the variousbiosynthetic processes (including growth) of the yeast cell. Thus, inyeast, there exists a fairly tight energetic coupling between theanabolic and catabolic processes. For practical purposes, thiswell-regulated or conservative type of glucose metabolism means thathigh cell densitites are required in order to achieve an acceptable rateof fermentation by non-proliferating yeast.

                  TABLE 16                                                        ______________________________________                                        Comparative steady-state fermentation                                         kinetics for yeast and Zymomonas                                              Physiological                                                                 State          Specific rate of glucose utilization                           sp. growth rate,                                                                             [gm glu/gm cell (DW)/hr]                                       u(hr.sup.-1)   Saccharomyces                                                                             Zymomonas                                          ______________________________________                                        Non-Growing (m.sub.e)                                                                        0.06*       1.5                                                u = O                                                                         Growing (q.sub.s)                                                             (a) Slowly     0.8*        3.0                                                u = 0.06                                                                      (b) Fast (u.sub.max)                                                                         1.7-2.1**   8.1-9.7                                            u = 0.4 (approx)                                                              ______________________________________                                         *Ref: J. Inst. Brew., 85, 342 (1979).                                         **Ref: Biotechnol. Letts., 1, 165 (1979) and Adv. in Biotechnol., 2, 195      (1981).                                                                       Note:                                                                         The specific rate of glucose utilization (q.sub.s) was determined in          steadystate chemostat culture as a function of the specific growth rate       (u) equivalent to the dilution rate and the value for the maintenance         energy coefficient (m.sub.e) was derived by extrapolation to u = o in         plots of q.sub.s versus D.                                               

In summary, the process of this invention makes it possible to carry outa continuous fermentation by Z. mobilis at increased specific rate ofsubstrate uptake and increased specific rate of product formation byregulating pH in the fermentation medium. The invention can be carriedout with or without nutrient limitation.

What is claimed is:
 1. A continuous process for the production ofethanol, which comprisesfeeding an aqueous substrate solutionsubstantially continuously to a reactor containing a fermentation mediumand a submerged culture of an organism of the genus Zymomonas;cultivating said organism to produce ethanol under substantially steadystate anaerobic conditions in an aqueous nutrient medium containingassimilable carbon, nitrogen and phosphorus; controlling pH in thefermentation medium between a pH of about 3.8 and a pH less than 4.5 sothat ethanol production is substantially uncoupled from cell growth andwherein the biomass expresses values for both the specific rate ofsubstrate uptake (q_(s)) and the specific rate of ethanol formation(q_(p)) which are greater than the values would be for a similar processconducted at a higher pH; and removing an effluent containing ethanolfrom the reactor.
 2. A process according to claim 1, which comprisescontrolling the pH between about 4.0 and about 4.2.
 3. A processaccording to claim 1, wherein fermentation is conducted at a temperatureof about 27° C. to about 37° C.
 4. A process according to claim 1,wherein fermentation is conducted at a temperature of about 33° C. toabout 37° C.
 5. A process according to claim 1, wherein said organism isa Z. mobilis strain selected from the group consisting of ATCC 10988,ATCC 29191, ATCC 31821 and ATCC 31823 or a Z. mobilis flocculent strainselected from the group consisting of ATCC 31822, ATCC 35001, ATCC 35000and NRRL B-12526.
 6. A process according to claim 1, wherein saidorganism is Z. mobilis strain ATCC
 29191. 7. A process according toclaim 1, wherein said substrate is comprised of glucose.
 8. A processaccording to claim 1, wherein fermentation is carried out in a fermenterwith cell recycle.
 9. A process according to claim 1, wherein ethanolproduction is accompanied by cell growth in an amount sufficient tomaintain a substantially constant biomass concentration in thefermentation medium.
 10. A process as claimed in claim 1, wherein the pHis controlled by adding a non-toxic, non-inhibiting hydroxide, organicacid or inorganic acid to the fermentation medium.
 11. A process asclaimed in claim 1, wherein a pH regulating agent selected from thegroup consisting of potassium hydroxide, sodium hydroxide andhydrochloric acid is added to the fermentation in an amount sufficientto control the pH.